Enzymatic production of difructose dianhydride IV from sucrose and relevant enzymes and genes coding for them

ABSTRACT

Disclosed is the production of difructose dianhydride IV from sugar. A sugar solution is subjected to reaction at room temperature or lower in an acidic buffer of pH 3.0-7.0 in the presence of a levansucrase derived from  Z. mobilis  to produce levan. The levansucrase is prepared by culturing  E. coli  BL21 (DE3)/pEL12 (KCTC 8661), harvesting and homogenizing the cells, and isolating levansucrase from the cell homogenate. Levan is purified from the reaction solution and subjected to reaction at 25-50° C. for 3-10 hours in an acidic buffer of pH 3.0-7.0 in the presence of a levan fructotransferase to produce difructose dianhydride IV. The levan fructotransferase is obtained from  E. coli  JUD81 (KCTC 0877BP). Also, disclosed are a gene coding for the levan fructotransferase and an expression vector pUDAF81 carrying the gene.

TECHNICAL FIELD

The present invention relates to the enzymatic production of difructosedianhydride IV (hereinafter referred to as “DFA IV”) from sucrose. Moreparticularly, the present invention relates to enzymes which take partin the production of DFA IV from sugar and their use. In addition, thepresent invention relates to the production of the intermediate productduring the production of DFA IV. Also, the present invention isconcerned with novel genes coding for the enzymes, expression vectorscarrying the genes, and transformed cells with the expression vectors.

BACKGROUND ART

Difructose dianhydride was first found in 1929 by Jackson et al. byanalyzing a by-product which was produced when inulin was treated withsulfuric acid to prepare a fructose syrup. Difructose dianhydride, akind of a cyclic disaccharide, consists of two fructose residues inwhich a reducing end of each residue is linked to a non-reducing hydroxygroup of the counter residue. There have been discovered five kinds ofdifructose dianhydride, named DFA I to DFA V, thus far. Of them, DFA IIand V are found to be synthesized only chemically while the others canbe produced enzymatically: DFA I and III are produced from inulin by theaction of inulin fructotransferase; and DFA IV from levan by the actionof levan fructotransferase.

Difructose dianhydride is a non-digestive, non-fermentativesub-saccharide which is not digested in the animal body. In addition tobeing useful as a low-calorie sweetener, the sub-saccharide plays a rolein inhibiting tooth decay and as a productive factor for Bifidusbacteria. It is also reported that difructose dianhydride is used as anabsorption factor of minerals in the body (Baik, B. H, Lee, Y. W., andLee, Y. B.; U.S. Pat. No. 5,700,832, UK. Pat. No. GB 2 308 547 A,Japanese Pat. Appl'n No. 8-51370, Korean Pat. Laid-Open Publication No.96-13376, Sakurai et al., 1997).

Representative of polyfructans, inulin and levan, both naturallyoccurring fructose homopolysaccharides, are used to prepare difructosedianhydride (Han, 1989). From them, difructose dianhydride can besynthesized chemically or enzymatically. Chemical synthesis ofdifructose dianhydride from the natural polyfructans, however, suffersfrom significant disadvantages. For example, chemical synthesis is oflow reaction selectivity, followed by complicated separation andpurification. What is worse, it produces pollution of the environment.Consequently, these problems do not vest economical production value inthe chemical synthesis. In contrast, enzymatic synthesis usingbio-catalysts, such as microbes or enzymes, is now regarded as beingvery economically favorable in synthesizing difructose dianhydride fromthe natural polyfructans.

Since the discovery of difructose dianhydride III synthase (inulinfructotransferase) from microbes by Tanaka in 1972, enzymes that havethe function of synthesizing difructose dianhydrides have been isolatedfrom several microbial sources. The difructose dianhydrides which can besynthesized by such microbe-derived enzymes include DFA I, III and IV.DFA IV is synthesized from levan by the catalytic action of levanfructotransferase and two microorganisms, Arthrobacter ureafaciens andArthrobacter nicotinovorans GS-9 are found to produce DFA IV.

Only a very small amount of levan fructotransferase is synthesized fromthese bacteria and, thus, its use in the production of difructosedianhydride is very unfavorable in terms of technical and economicalaspects. Now generally, in order to obtain a large amount of a gene ofinterest, a gene recombinant technique is employed. That is, a levanfructotransferase gene is first isolated from its microbial source andcloned, followed by mass-expression in E. coli. Of the levanfructotransferase-producing strains discovered so far, only A.nicotinovorans GS-9 is achieved in cloning its levan fructotransferasegene.

Being a substrate of levan fructotransferase to produce DFA IV, levan, ahomopolysaccharide of fructose, is prepared from sucrose by thetransfructosylation reaction of levansucrase. The enzymes which cancatalyze the hydrolysis of sucrose are exemplified by sucrase(beta-D-fructofuranosidase, EC 3.2.1.26), levanase (beta-2,6-D-fructanfructanohydrolase, EC 3.2.1.65), levansucrase (beta-2,6-fructan:D-glucose-1-fructosyl transferase, EC 2.4.1.10), maltase(alpha-D-glucoside glucohydrolase, EC 3.2.1.20), etc. Levan can beproduced from sucrose by taking advantage of the transfructosylationactivity of levansucrase (Tanaka & Yamamoto, J. Biochem., 85, 287(1979)).

There are disclosed methods for the production of levan usinglevansucrase. For example, it is described in U.S. Pat. No. 4,879,228and International Publication No. WO 86-4091 that levan is produced by afermentation process in which advantage is taken of the microbesemploying sucrose in their metabolism. The patents, however, suffer frommany problems. There is required a lengthy culture period to producelevan. Further, because the culture contains various products, adifficult purification procedure is needed, giving rise to a decrease inthe production yield of levan. When account is taken of these problems,an enzyme reaction process has an advantage over the culture process inthat, because the products of levansucrase are dependent on the reactionconditions for the production of levan from sucrose, desirable productscan be obtained by controlling the conditions with ease.

It is reported that levan finds numerous applications in the medicinalfield, such as a serum substituent (Dedonder et al., Bull. Soc. Chim.Biol., 39, 438 (1957); Schechter et al., J. Lab. Clin. Med., 61,962(1963)), a colloid stabilizer, an immune agent, a pharmacologicalenhancer, etc. (Leibovici et al., Anticancer Res., 5, 553 (1985); Starket al., Br. J. Exp. Path., 67, 141 (1986)), the foodstuffs field, suchas quality improver, a stabilizer, an additive for health food, etc.(Hatcher et al., Bioprocess, Technol., 11, 1(1989)), the typographicfield, and the cosmetic field (Whiting et al., J. Inst. Brew., 73, 422(1967); Han, Bioprocess Technol., 12, 1(1920)).

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide an enzymatic processfor producing levan from sugar at a high yield.

It is another object of the present invention to provide a levansucrasegene derived from Z. mobilis, which contains such a specific tool as toallow its protein to be purified with ease.

It is a further object of the present invention to provide a recombinantexpression vector carrying a levansucrase gene derived from Z. mobilis,which can express levansucrase in E. coli.

It is still a further object of the present invention to provide abacterial species which is transformed by the recombinant expressionvector and can produce levansucrase.

It is yet a further object of the present invention to provide a processof preparing levansucrase by taking advantage of the transformedbacteria.

It is still another object of the present invention to provide anenzymatic process for producing DFA IV from levan at a high yield.

It is yet another object of the present invention to provide a novelbacteria A. ureafaciens K2032, which produces levan fructotransferase.

It is yet another object of the present invention to provide a novelgene coding for levan fructotransferase, derived from A. ureafaciensK2032.

It is yet another object of the present invention to provide arecombinant levan fructotransferase gene, which contains such a specifictool as to allow its protein to be purified with ease.

It is yet another object of the present invention to provide arecombinant expression vector carrying the recombinant gene and abacterial species which anchors the recombinant expression vector.

It is yet another object of the present invention to provide a processof preparing levan fructotransferase by taking advantage of thetransformed bacteria.

It is yet another object of the present invention to provide anenzymatic process for producing DFA IV from sucrose at a high yield byutilizing the levansucrase and the levan fructotransferase.

In accordance with a first embodiment of the present invention, there isprovided a process for producing levan from sugar, in which a sugarsolution is subjected to reaction at room temperature or lower in anacidic buffer of pH 3.0-7.0 in the presence of a levansucrase derivedfrom Z. mobilis. In one version of this embodiment, the reaction iscarried out at 0-15° C. and the sugar solution has a sugar concentrationof 10-30% (w/v).

In accordance with a fourth embodiment of the present invention, thereis provided a recombinant expression vector, carrying the levansucrasegene.

In accordance with a fifth embodiment of the present invention, there isprovided a novel bacteria E. coli BL21 (DE3)/pEL12 (KCTC 8661), in whichthe plasmid carries a levansucrase gene derived from Z. mobilis.

In accordance with a sixth embodiment of the present invention, there isprovided a process of preparing levansucrase, comprising the steps ofculturing a bacterial species anchoring a levansucrase gene-carrying,expression plasmid, harvesting and homogenizing the cells, and isolatinglevansucrase from the cell homogenate. In one version of thisembodiment, the levansucrase gene has a base sequence stretch encodinghistidine residues at its 5′- or 3′-end. In another version of thisembodiment, the isolating step is carried out using metal ion-affinitychromatography and the bacterial species is E. coli BL21(DE3)/pEL12(KCTC 8661P).

In accordance with a seventh embodiment of the present invention, thereis provided a novel microorganism A. ureafaciens K2032, which shows anactivity of selectively producing difructose dianhydride IV from levanand an activity of degrading levan.

In accordance with an eighth embodiment of the present invention, thereis provided a novel levan fructotransferase of SEQ ID. NO: 1.

In accordance with a ninth embodiment of the present invention, there isprovided a novel levan fructotransferase polynucleotide of SEQ ID NO: 2.

In accordance with a tenth embodiment of the present invention, there isprovided a novel levan fructotransferase of SEQ ID NO: 1 encoded by apolynucleotide of SEQ ID NO: 2, as provided in the compositepolynucleotide/amino acid sequence of SEQ ID NO: 3.

In accordance with an eleventh embodiment of the present invention,there is provided a recombinant expression vector, carrying the levanfructotransferase gene.

In accordance with a twelfth embodiment of the present invention, thereis provided a process for producing DFA IV from levan, in which a levansolution is subjected to reaction at 25-50° C. for 3-10 hours in anacidic buffer of pH 3.0-7.0 in the presence of a levanfructotransferase. In one version of this embodiment, the reaction iscarried out at 37° C. In another version of this embodiment, the acidicbuffer is a phosphate buffer of pH 5.8 and the levan solution has alevan concentration of 5-15% (w/v).

In accordance with a thirteenth embodiment of the present invention,there is provided a process of preparing levan fructotransferase,comprising the steps of culturing a bacterial species anchoring a levanfructotransferase gene-carrying, expression plasmid, harvesting andhomogenizing the cells, and isolating levan fructotransferase from thecell homogenate. In one version of this embodiment, the levanfructotransferase has histidine residues at its N- or C-terminus and theisolating step is carried out using metal ion-affinity chromatography.

In accordance with an fourteenth embodiment of the present invention,there is provided a process for producing difructose dianhydride IV fromsucrose, comprising the steps of reacting a sugar solution at roomtemperature or lower in an acidic buffer of pH 3.0-7.0 in the presenceof a levansucrase to produce levan, purifying the levan from the sugarreaction mixture, partially or completely, reacting a levan solution at25-50° C. for 3-10 hours in an acidic buffer of pH 3.0-7.0 in thepresence of a levan fructotransferase to produce difructose dianhydrideIV, and isolating the difructose dianhydride IV from the levan reactionmixture.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

FIG. 1 shows the various steps in a process for the production ofdifructose dianhydride IV from sucrose.

FIG. 2 shows the restriction map of plasmid pZL8, in which B stand forBamHI, H for HindIII, E for EcoRV, N for NcoI, A for AseI, S for SphI,(B::Sa) for BamHI::Sau3A1.

FIG. 3 shows a base sequence of the levansucrase gene (SEQ ID NO. 5) andan amino acid sequence deduced therefrom (SEQ ID NO. 6).

FIG. 4 shows a SDS-PAGE result of purified levansucrase, in which astandard protein is electrophoresed in lane M, total protein of E. coliin lane 1 and the purified levansucrase in lane 2.

FIG. 5 shows a SDS-PAGE result of purified levan fructotransferase, inwhich a standard protein is electrophoresed in lane 1 and the purifiedlevan fructotransferase in lane 2.

FIG. 6 shows a nucleotide base sequence of the levan fructotransferasegene (SEQ ID NO: 2) and an amino acid sequence (SEQ ID NO: 1) deducedtherefrom, with translation start and restriction sites indicated.

FIG. 7 shows a structure of the recombinant expression vector pUDFA18carrying the levan fructotransferase gene, in which B stand for BamHI, Pfor PstI, N for NcoI, S for SalI, C for ClaI, and (N/A) for NcoI/AfIII.

FIG. 8 shows the total protein pattern of E. coli JUD81 in SDS-PAGE inwhich a bacterial protein is electrophoresed in lane 1 and a standardprotein in lane 2.

FIG. 9 shows the change of the amounts of DFA IV and other saccharidesin the reaction mixture in accordance with the reaction time period.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention pertains to the enzymatic production of DFA IVfrom sucrose, which is achieved by carrying out the following processsteps: a levan production process by levansucrase, a purification andconcentration process of levan, a DFA IV production process by levanfructotransferase, and a DFA IV recovery process by crystallization.Below, a description will be given, in detail, of these processes (FIG.1).

1. Levan Production Process by Levansucrase

In accordance with the present invention, the production of levan isbased on the catalysis of the levansucrase isolated from Z. mobilis withsucrose serving as a substrate. In the presence of the levansucrase,sucrose is subjected to reaction at room temperature, preferably at0-15° C. and more preferably at 10° C. for 20-80 hours and preferablyfor 20 hours in an acidic buffer solution of pH 3.0-7.0 and preferablyof pH 5.0. Therefore, most preferable is to react the sucrose at 10° C.for 20 hours in an acetic acid buffer of pH 5.0.

As for the substrate, its examples include refined sugar and raw sugarwith preference to refined sugar.

The sugar preferably has a concentration of 10-30% (w/v) based on thetotal volume of the culture and most preferably 20% (w/v).

Regardless of whether it is natural or is made by genetic recombination,the levansucrase that is derived from Zymomonas mobilis is useful in thepresent invention. For the effective catalysis of levan production, theenzyme is used at an amount of 0.42-3.0 U/ml based on the total volumeof the reaction solution, and preferably at an amount of 2.08 U/ml.

Usually, gene manipulation leads to the mass production of proteins,which is very advantageous in purification. Thus, the levansucrase ofthe present invention is preferably obtained through geneticrecombination. To this end, first, a plasmid carrying a Z.mobilis-derived levansucrase gene, e.g., the plasmid pZL8 obtained fromE. coli KCTC 8546P, is used as a template to amplify the levansucrasegene by the polymerase chain reaction (PCR) with appropriate syntheticprimers. Subsequently, the amplified gene of interest is inserted in avector which is able to be expressed in a suitable host. For instance,when E. coli is used as a host, the levansucrase gene amplified isinserted in pET3d (Stratagene) to construct an E. coli-expressionplasmid, e.g., pEL11 which is then transformed into the host. Thistransformed host cell is cultured under an appropriate condition andhomogenized, followed by centrifugation. To the supernatant, ammoniumsulfate is added to give crude levansucrase.

Since the crude enzyme contains various proteins in addition tolevansucrase, a complicated purification procedure is required to obtainpure levansucrase. In order to overcome this problem, there is provideda levansucrase further comprising histidine residues at its N- orC-terminus, which can be simply purified by metal ion-exchange columnchromatography, in accordance with the present invention.

Histidine residues can be attached to the N- or C-end of levansucrase asfollows. First, PCR is conducted using a set of synthetic primers whichare designed to have a histidine base sequence at their 3′ or 5′-endwhile a plasmid carrying a Z. mobilis-derived levansucrase gene, e.g.,the plasmid pZL8 obtained from E. coli KCTC 8546P is used as a template,so as to yield a large quantity of a levansucrase gene which furthercomprises a nucleotide sequence coding histidine at its 3′ or 5′-end.Thereafter, the amplified gene is inserted in a vector which isexpressable in a suitable host. For instance, when E. coli is used as ahost, the levansucrase gene amplified is inserted in pET3d to constructan E. coli-expression plasmid, e.g., pEL11 which is then transformedinto the host. This transformed host cell is cultured under anappropriate condition and homogenized, followed by purification withmetal ion-exchange column chromatography to obtain pure levansucrase.

Experimental data show that, when 0.5-2 U of the levansucrase purifiedis added in a 20% sugar solution, levan is produced at an amount of 50g/l after 10 hours of the enzymatic reaction at 10° C.

By using the levansucrase, levan, a water-soluble, homo-polysaccharide,can be mass-produced in a batch process or a continuous process at ahigh purity (≧98%) at a high yield (35-40% from sucrose). In the case ofa batch process, because the activity of the levansucrase is inhibitedby glucose, a product of the enzyme reaction, the concentration of thesubstrate must be controlled. Preferable is a 20% substrate solution. Asfor the continuous process, there is needed a reactor, e.g., a membranebioreactor, equipped with a means of continuously removing the glucoseproduced.

After the production of levan, to discard the enzyme used is tooextravagant. In this regard, the enzyme may be immobilized to a suitablematrix, such as hydroxyapatite, iron-bead, non-porous glass, wire, etc.in order to reuse the enzyme.

In addition to being a substrate for producing DFA IV, the levanproduced according to the present invention, as aforementioned, can beused for various purposes, such as a material for foodstuffs andmedicines.

2. Purification and Concentration Process of Levan

After completion of the enzymatic production reaction of levan, theresulting levan-containing solution must be deprived of unreactedresidual sucrose, glucose, and oligosaccharides, followed byconcentration preferably by as much as 10-15%. To this end, availableare various techniques, such as solvent precipitation, ultrafiltration,diafiltration, reverse osmosis, chromatography, etc. For a small scale,such as a laboratory scale, an organic solvent method using, forexample, ethanol is suitable. Only to precipitate twice or three timesin the organic solvent is sufficient to obtain pure levan. For a largescale, a membrane filtration process is preferably employed, as invarious polymer production processes.

Where the levan is produced in a continuous process, membrane isolationtechniques, such as microfiltration (MF), ultrafiltration (UF) orreverse osmosis (RO), are used to effectively isolate the product ofinterest or remove by-products from the reaction solution. Thesetechniques have advantages of being able to be operated at lowtemperatures as well as allow the simple measurement of energy yield andseparation capacity. For example, the glucose can be removed from thereaction mixture with the aid of ultrafiltration (UHF-500-E-90A, 500,000molecular weight cutoff, A/G Technology Co.) or MF (CPP-1-k-9A, 0.1microl, A/G Technology co.). Preferred is UHF-500-E-90A, which issuperior in permeate flux and permeability (permeate flux, 376.47ml.ft²; permeability, 21.16 ml/min/ft²/psi; 500,000 nominal molecularweight cutoff)

To achieve excellent purification of the levan, the filtration describedabove may be used in combination with the organic solvent precipitation.Alternatively, the purification of the levan may be accomplished byconducting various membrane isolation techniques in multiple steps.

3. DFA IV Production Process by Levan Fructotransferase

In accordance with the present invention, DFA IV is produced frompartially purified or pure levan by the catalysis of levanfructotransferase.

In the presence of the levan fructotransferase, levan is subjected toreaction at 25-50° C. and preferably at 37° C. for a time period of 3-10hours and preferably for 5 hours in a buffer of pH 3.0-7.0 andpreferably of pH 5.8. Preferable is a phosphate buffer.

Useful to produce difructose dianhydride is partially or completelypurified levan with preference to completely purified levan.

In the reaction solution, the levan is preferably controlled to have aconcentration of 5-15% (w/v) based on the total volume of the cultureand most preferably 10% (w/v).

Regardless of whether it is natural or is made by genetic recombinationfrom transformable microbes such as E. coli or yeasts, the levanfructotransferase that is derived from A. ureafaciens is useful in thepresent invention.

The levan fructotransferase of the present invention is preferablyobtained through genetic recombination because gene manipulation leadsto the mass production of the protein, which is very advantageous inpurification. To this end, first, a plasmid carrying an A.ureafaciens-derived levan fructotransferase gene, e.g., the plasmidpDA18, is used as a template to amplify the levan fructotransferase geneby PCR with appropriate synthetic primers. Subsequently, the amplifiedgene of interest is inserted in a vector which is able to be expressedin a suitable host, such as bacteria or yeast.

In accordance with the present invention, the levan fructotransferasegene amplified is inserted in pUC18 to construct an E. coli-expressionplasmid pUDFA18, which is then transformed into E. coli DH5α. The newbacterial cell thus obtained was named E. coli JUD81 and deposited inKorean Collection for Type Cultures, Korean Research Institute ofBioscience and Biotechnology on Sep. 1, 1999 at accession No. KCTC8961P. The original deposit was converted to a deposit under theBudapest Treaty on Oct. 19, 2000 at accession No. KCTC 0877BP. Thistransformed host cell is cultured under an appropriate condition andhomogenized, followed by centrifugation. Addition of ammonium sulfate tothe supernatant allows the production of crude levan fructotransferase.

Since the crude enzyme contains not only levan fructotransferase, butalso various other proteins, the crude enzyme must undergo a complicatedpurification procedure to obtain only levan fructotransferase. In orderto overcome this problem, there is provided a levan fructotransferasefurther comprising histidine residues at its N- or C-terminus, which canbe simply purified by metal ion-exchange column chromatography, inaccordance with the present invention.

Histidine residues can be attached to the N- or C-end of levanfructotransferase as follows. First, PCR is conducted using a set ofsynthetic primers which are designed to have a histidine base sequenceat their 3′ or 5′-end while a plasmid carrying an A. ureafaciens-derivedlevan fructotransferase gene, e.g., the plasmid pUDFA18 obtained from E.coli KCTC 0877BP is used as a template, so as to yield a large quantityof a levan fructotransferase gene which further comprises a nucleotidesequence coding histidine at its 3′ or 5′-end. Thereafter, the amplifiedgene is inserted in a vector which is expressable in a suitable host.For instance, when E. coli is used as a host, the levanfructotransferase gene amplified is inserted in pUC18 to construct an E.coli-expression plasmid, e.g., pUDFA18 with which the host is thentransformed. This transformed host cell is cultured under an appropriatecondition and homogenized, followed by the purification with metalion-exchange column chromatography to isolate the levanfructotransferase comprising histidine residues at its N- or C-terminus.

Like levan, DFA IV can be mass-produced in a batch process or acontinuous process by using the levan fructotransferase. This enzyme isalso immobilized to a suitable matrix, such as hydroxyapatite,iron-bead, non-porous glass, wire, etc. in order to reuse the enzyme.

Experimental data show that, when 20 g of the levan produced by thecatalysis of the levansucrase of Z. mobilis was allowed to react with ahomogenized cell solution containing 25 U of the levan fructotransferaseat 37° C. in 1000 ml of a phosphate buffer (pH 5.8), DFA IV was producedat a yield of 60% after 20 hours of the reaction.

4. DFA IV Recovery Process by Crystallization

From the levan produced by the catalysis of the levansucrase of Z.mobilis, a reaction solution comprising DFA IV 60%, fructose 5%, limitedlevan 30%, and other saccharides (mainly, oligosaccharides) 5% isobtained as a result of the decomposition activity of the levanfructotransferase. This reaction solution is subjected to a purificationprocess to isolate DFA IV, or concentrated to some degree to give asolution with a high content of DFA IV. If necessary, the reactionsolution itself may be used instead of DFA IV.

Isolation of DFA IV from the resulting reaction solution may be achievedby conducting crystallization, solvent precipitation, diafiltration,ultrafiltration, reverse osmosis, evaporation, drying, chromatography,etc., alone or in combination. Useful in the present invention is amembrane filtration, which is usually used in various polymer productionprocesses, in practice.

In addition to being more stable to heat than sucrose, DFA IV wellcrystallizes. For the purpose of crystallizing DFA IV, the enzymolyzedsolution of levan needs to be concentrated. In this connection, theabove-mentioned filtration processes or a popularized standard sugarsolution evaporation process may be utilized. Alternatively, theenzymolyzed solution may be primarily concentrated to some degree byfiltration and then, heated at 70-80° C. until DFA IV reaches adesirable concentration. At this time, a heating temperature exceeding100° C. causes caramelization. The DFA IV filtrate of the primaryfiltration may be used as a diluting solution of the levan.

In a small scale site, such as a laboratory, the DFA IV solution ispreferably added with two volumes of an organic solvent, such asethanol, to precipitate oligosaccharides or limited levan and theresulting supernatant is concentrated using a rotary evaporator, afterwhich the concentrate is controlled to an ethanol concentration of 95%with 100% ethanol and allowed to stand at 4° C. to induce crystalprecipitation. Alternatively, while a supersaturated solution of DFA IVis allowed to stand at 4° C. to induce natural crystallization, DFA IVcrystals are seeded to rapidly form crystals. These batchcrystallization processes are, however, disadvantageous in that it isdifficult to obtain uniform crystals. In contrast, a continuous processis very useful in crystallizing DFA IV at a uniform size and shape aswell as has an advantage of being of low cost compared with the batchprocess.

A better understanding of the present invention may be obtained in lightof the following examples which are set forth to illustrate, but are notto be construed to limit the present invention.

EXAMPLE Experimental Example 1

Cloning of Levansucrase Gene

The isolation of genomic DNA from Z. mobilis was executed according tothe instruction of Raymond and Tate (Raymond and Tate, Recombinant DNAtechniques, p.162, 1983, Addison-Wesley Publ.). After being washed witha TEN buffer (10 mM Tris-Cl pH 7.6, 1 mM EDTA, 10 mM NaCl), 1 g of amass of Zymomonas mobilis ZM1 (ATCC 10988) which had been cultured in aYPS medium (Yeast extract 0.5%, peptone 1%, sucrose 2%) was suspended in10 ml of an SET buffer (sucrose 20%, 50 mM Tris-Cl pH 7.6, 50 mM EDTA)and treated with lysozyme (Sigma, 5 mg/ml PEN buffer). After 30 min ofthe enzyme treatment, the mixture was added with 10 ml of a TEN bufferand 1 ml of 10% sodium dodecylsulfate (SDS) and slowly agitated. To thelysed cells was added 2 ml of a 5 M NaCl solution and 20 ml of a TENbuffer and the resulting solution was added with an equal volume of aphenol solution saturated with a TE buffer (10 mM Tris-HCl pH 7.6, 1 mMEDTA) and agitated for 5 min, followed by centrifugation at 10,000 rpmfor 5 min. The supernatant was further centrifuged after being wellmixed with chloroform/n-amyl alcohol (24:1). Addition of two volumes ofcold ethanol to the resulting supernatant precipitated DNA.

After purification, the Z. mobilis genomic DNA was partially cut withthe restriction enzyme Sau3AI and DNA fragments in a size range of 4-10kb were isolated with the aid of 1% agarose gel. Separately, pUC119(Takara, Japan) was cut with the restriction enzyme BamHI anddephosphorylated. Recombinant plasmids were obtained by ligating theisolated DNA fragments of 4-10 kb to digested pUC 119.

Following the techniques of Mandel and Higa (Mandel and Higa, J. Mol.Biol. 53, 159 (1970)) and Inoue et al. (Inoue et al., Gene 96, 23(1990)), transformation was executed. First, E. coli JM109 that had beencultured at 18° C. for 36 hours in 30 ml of an SOB medium (Trypton 2%,Yeast extract 0.5%, 10 mM NaCl, 2.5 mM CaCl₂, 10 mM MgCl₂, 10 mM MgSO₄)was kept in ice for 10 min and centrifuged at 2500×g for 10 min, afterwhich the cell pellet was suspended in 10 ml of a TB solution (10 mMPIPES, 55 mM MnCl₂, 250 mM KCl). The resulting suspension was added withdimethyl sulfoxide (DMSO) to the final concentration of 7%, kept in icefor 10 min, aliquoted at 400 μl in cold tubes, and stored in a liquidnitrogen tank. For transformation, the frozen cell suspension was thawedat room temperature and well mixed with 10 μl of the recombinant DNA(DNA 1 μg) and this mixture was refrigerated for 30 min and subjected toheat-shock at 42° C. for 30 sec. Subsequently, the transformationsolution was added with 800 μl of an SOC medium (Trypton 2%, Yeastextract 0.5%, 10 mM NaCl, 2.5 mM CaCl₂, 10 mM MgCl₂, 10 mM MgSO₄, 10 mMGlucose), vigorously shaken at 37° C. for 1 hour, spread over thesurface of an LB agar plate containing ampicillin and chloramphenicol,and cultured at 37° C. for 16 hours.

The colonies formed were transferred to an MG agar plate supplementedwith sucrose (20 g/l) and ampicillin (50 mg/ml) and cultured at 37° C.for 2 days. Spraying GOD-PAP (Böeringer Mannheim) onto the plate allowedthe selection of 13 colonies which formed red halos around themselves.Further selection was on the colonies which could be of sucrase activityas well as of levan formativity as measured according to the instructionof Gay et al. (Gay et al., J. Bacteriol. 153, 1432(1983)). From the E.Coli colonies, plasmids were isolated and named “pZL8”. E. coli DH5αbearing the plasmid was deposited in Korean Collection for TypeCultures, Korean Research Institute of Bioscience and Biotechnology onDec. 14, 1993 at deposition No. KCTC 8546P.

Gene Identification by Levan Formation

Cultivation of the E. coli bearing pZL8 at 37° C. for 3 days in an LBSbroth (LB broth supplemented with sucrose at 5%) resulted in thedetection of polymers similar to those of levan. The culture wascentrifuged and the supernatant was added with three volumes of coldethanol to precipitate polymers. They were dissolved in distilled waterand precipitated with ethanol. This ethanol precipitation procedure wascarried out twice more. The raw polymers thus obtained were separated bythin layer chromatography (TLC) on silica gel developing withn-butanol/pyridine/water (8/1/1), followed by the coloration with a 1%anisaldehyde-sulfuric acid solution.

Because levan, a macro-molecule with a molecular weight of 5×10⁷Daltons, cannot move on TLC, it was hydrolyzed according to theinstruction of Tanaka (Tanaka et al., J. Biochem. 90, 521 (1981)). 100μl of the raw polymer solution was boiled for 15 min, along with 50 μlof 2.5% oxalic acid, and the hydrolyzed solution was subjected to TLC.The acid hydrolysate was extended for 15 min, only fructose wasobserved. There were difficulties in analyzing the sugar components withaccuracy by TLC because the Rf value was 0.46 for glucose, 0.52 forfructose, and 0.4 for sucrose. To compensate for this, HPLC analysis wasexecuted.

20 μl of each of the hydrolysate samples was filtered through a 0.2 μmmembrane and subjected to HPLC with the aid of an HPLC apparatus, suchas sold by Waters, identified as “Model R401”, by flowing water at arate of 0.6 ml/min through a column carbohydrate HPX-87C (Bio-Rad).Detection was conducted using RI (Oven Temp. 85-90° C.). All of thesamples showed two peaks: a peak was read at 4.19 min for oxalic acidand the other peak at 9.29 min for sucrose. These data, therefore,demonstrated that the polymer obtained above was composed of fructoseresidues. In addition, no inulin (β(2-1) linked polyfructan) formativeactivity was observed in Z. mobilis (Toran et al., J. Biotechnol. Lett.7, 527(1985)). Consequently, it was made certain that the polymersformed in the E. coli bacteria bearing the plasmid pZL8 were levan(β(2-6) linked polyfructan) (Viikari, L., CRC Critical Reviews inBiotechnology 7, 237 (1988)) and a levansucrase gene was introduced inthe plasmid pZL8.

Gene Identification by Gene Mapping

The recombinant plasmid obtained above was digested with variousrestriction enzymes and electrophoresed on 1% agarose gel. Based on theDNA fragment data on the agarose gel, a restriction enzyme map of theplasmid pZL8 was drawn, providing the information that the genomic DNAintroduced in the plasmid pZL8 was 4.5 kb long.

To be used as a probe, the plasmid pZL8 was labeled with a DNA labelingkit (Böeringer Mannheim). Separately, the Zymomonas mobilis genomic DNAprepared above was cut with Restriction enzymes, EcoRI, EcoRV, HindIIIand NcoI and electrophoresed on 1% agarose gel. Then, the genomic DNAfragments on the agarose gel were transferred to a nylon membrane andhybridized with the probe in accordance with the teaching of Southern(Southern, J. Mol. Biol., 98, 503 (1975)). Referring to the instructionenclosed in the DNA labeling kit, coloration was achieved; Because theplasmid pZL8 has a unique restriction enzyme site for each of EcoRV,HindIII and NcoI, the DNA sequence of 4.5 kb introduced into the plasmidpZL8 must be from Z. mobilis.

Gene Identification by Base Sequencing

An examination was made of the levansucrase activity of the mutantbacteria bearing delete pZL8 which was made by use of restrictionenzymes. From the examination, it was recognized that the levansucrasegene was positioned in the 1.6 kb base sequence between the EcoRV siteand the AseI site, as shown in FIG. 2.

Delete plasmids, which were constructed from the 1.6 kb DNA fragmentswith the aid of an exo/Mungbean delete kit (Takara, Japan), wereamplified. 2 μl of the prepared plasmids prepared from the mutant cellswas denatured with 2 N NaOH, neutralized with ammonium acetate (pH 4.5),allowed to precipitate by ethanol, and dissolved in deionized water.Using a Sequenase kit (USB, U.S.A.), the denatured plasmids were labeledand 4 μl of the reaction was boiled for 3 min and electrophoresed on 8%polyacrylamide-8M urea gel at 1500-1700 V for 3 hours. The gel was driedand exposed to X-ray film at −70° C. for about 8 hours. The reading ofthe base sequence which appeared on the sensitized X-ray film revealedthat the total levansucrase gene including its one termination codon is1800 bp long: the structural gene corresponding to the amino acidsequence is 1269 bp long. The base sequence of the levansucrase gene andthe amino acid sequence deduced therefrom are as shown in FIG. 3.

Gene Identification by Amino Acid Sequencing Z. mobilis ZM1 was culturedat 30° C. for 24 hours in a YPS medium and the multiplied cells wereharvested, suspended in a 20 mM phosphate buffer (pH 6.8), shaken at 30°C. for 15 min, and centrifuged according to the introduction of Yanase(Yanase et al., Biosci. Biotech. Biochem. 56, 1335 (1992)). Thesupernatant was used as a crude enzyme solution.

In this crude solution, ammonium sulfate was saturated up to 50%, toprecipitate proteins which were recovered by centrifugation at 8,000×gfor 20 min. The protein mass was dissolved in a 0.02 M phosphate buffer(pH 6.8), followed by dialysis in the same buffer. In this regard,elution was conducted at a rate of 0.5 ml/min through a column (2.5×10cm) charged with a weak anion exchange resin (DEAE-Toyopearl 650M). In alinear concentration gradient of NaCl from 0 to 0.5 M, the eluate at 0.3M was collected. The eluate was concentrated and purified followed byHydroxyapatite column chromatography. After being concentrated, Theprotein was allowed to precipitate with 20% saturated ammonium sulfate.And finally the concentrate was loaded on a gel filtration column(Superose 12, Pharmacia) to elute a fraction containing a molecularweight of 91,000. The final purification yield was 18.3 fold of thecrude enzyme from Z. mobilis, with 16.5% of the enzyme recovered in thepreparation step (Table 1). The solution was used as a levansucrasesolution.

TABLE 1 Summary of levansucrase purification steps from Z. mobilis Spec.Volume U Protein Act. Yield Purifi. Step (ml) total (mg/ml) (U/mg) (%)Fold Cell washed 1,300 -a 0.35 — — — 1st (NH₄)₂SO₄ 115 — 1.28 — — —Ion-exchange 38 4.35 0.57 0.21 100 1.00 Hydroxyapatite 20 2.58 0.41 0.3165 1.52 2nd (NH₄)₂SO₄ 2 0.96 0.46 1.04 21 5.07 Superose 12 1.5 0.72 0.133.75 16.5 18.3 a: could not be determined.

With the aid of a protein-peptide sequencing system (Applied Biosystems,Model 477A), the amino acid sequence of the purified levansucrase wasdetermined at its N-terminal in the Edman degradation procedure. As aresult, a stretch of seven amino acid residues,Met-Leu-Asn-Lys-Ala-Gly-Ile, was sequenced, reflecting the correspondingbase sequence of the DNA. In particular, the levU gene was revealed tohave no base sequences which correspond to the signal peptides, whichare usually found in secretory proteins. The nucleotide and amino acidsequences of the levansucrase gene from Z. mobilis was registered in theGenBank, U.S.A. (Accession No. AF081588).

Experimental Example 2 Production of Levansucrase

Step 1 Construction of Expression Vector

E. coli KCTC 8546P was cultured at 37° C. overnight in an LB medium(yeast extract 0.5%, trypton 1%, NaCl 1%), after which plasmid pZL8 wasextracted from the culture, according to the method of Maniatis et al.(Maniatis et al., Molecular Cloning: A Laboratory manual, 2nd ed., ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., U.S.A.).

A levansucrase gene 1296 bp long was amplified by a PCR which employedtwo primers with the plasmid pZL 8 serving as a template.

A reaction mixture comprising a 10×Taq polymerase buffer (10 ul), a10×dNTP mix (10 ul) (dATP, dCTP, dGTP, and dTTP, 2 mM each), the primers1 and 2 (1 ul (100 pM), each), the plasmid pZL8 DNA (5 ul (20 ng)), anddeionized water (72 ul) was thermally treated, along with a mineral oil(100 ul), at 95° C. for 5 min to denature the DNA. The reaction mixturewas added with a Taq polymerase (1 ul (5 U)) after being cooled to 72°C. and then, allowed to undergo 25 thermal cycles in which heatingprocesses were performed in the order of at 95° C. for 1 min, at 55° C.for 2 min and at 72° C. for 3 min.

The PCR product thus obtained was digested with the restriction enzymeAflIII and introduced into the NcoI site of the plasmid pET3d(Stratagene) to construct the E. coli-expression plasmid pEL11.

Step 2 Transformation of E. coli

E. coli DH5α and BL21 (DE3) were separately cultured at 18° C. for 36hours in 30 ml of an SOB medium (Trypton 2%, Yeast extract 0.5%, NaCl 10mM, calcium chloride 2.5 mM, magnesium chloride 10 mM, magnesium sulfate10 mM), followed by centrifugation to harvest the bacteria.Subsequently, the cultured bacteria were separately suspended in 10 mlof a TB buffer (Pipes 10 mM, manganese chloride 55 mM, calcium chloride15 mM, potassium chloride 250 mM), followed by centrifugation to collectthe cells. The cell mass was resuspended in 12 ml of a TB buffer andafter being added with DMSO (dimethyl sulfoxide) at a concentration of7%, the suspension was kept on ice and stored at an aliquot of 400 ul ina liquid nitrogen tank.

After being thawed, the E. coli DH5α was added with 10 ul of the plasmidpEL11 constructed in the Step 1 and kept on ice for 30 min, followed byheat shock at 42° C. for 30 sec for transformation. The transformed cellmixture was added with 800 ul of an SOC medium, vigorously agitated at37° C. for 60 min, and spread on an LB agar containing 170 mg/ml ofampicillin, which was then incubated at 37° C. for 16 hours. Coloniesformed on the agar medium were taken and cultured in LB brothscontaining ampicillin. Plasmids were extracted from the cells culturedand tested for whether they carried the levansucrase gene, byrestriction enzyme mapping.

E. coli BL21 (DE3) was transformed by the recombinant plasmid which hadbeen ascertained to carry the levansucrase gene, spread on anampicillin-added LB agar, and incubated at 37° C.

Step 3 Production and Isolation of Levansucrase

The transformed cells were inoculated in M9-ZB media (Trypton 10 g, NaCl5 g, NH₄Cl 1 g, KH₂PO₄ 3 g, Na₂HPO₄ 6 g, Glucose 4 g, 1 M MgSO₄1 ml) andcultured with agitating. When the absorbance at 600 nm of the culturereached 0.7 during the cultivation, IPTG (Sigma) was added to theconcentration of 1 mM with the aim of inducing the expression of thelevansucrase.

To the cell pellet which was obtained by centrifuging the culture, a 100mM Tris buffer (pH 7.0) was added at an amount of {fraction (1/10)}volume of the culture, and the suspended cells were homogenated bysonicating three times for 30 sec with the aid of a sonifier (Branson),followed by centrifugation at 12000×g for 60 min. The supernatant thusobtained was added with ammonium sulfate to the concentration of 20% andcentrifuged to give above 94%-pure levansucrase. This crude enzyme masswas found to have a levansucrase activity of 7.8 U/ml, if harvested at 4hours after the IPTG induction, as measured according to the instructiondescribed in Song et al. (song and Rhee, Biotechnol. Lett. 16, 1305(1994)) Example 2. It amounted to 30% of the total quantity of theproteins produced from the E. coli.

Comparative Example 1

Z. mobilis ATCC 10988 was cultured in a YPS medium (Yeast extract 1%,potassium phosphate 0.1%, sucrose 20%) at 30° C. for 18 hours, and fromthe culture was obtained levansucrase which was measured to have anactivity of 1.5 U/ml.

Therefore, the amount of the levansucrase produced from the transformedcells in Experimental Example 2 was 5.2 times as much as that of thelevansucrase produced from Z. mobilis ATCC 10988.

Example 1

While E. coli was cultured as in the Step 3 of Experimental Example 2,the culture was taken out at 10 ml at regular intervals of time, andcentrifuged. To the cell pellet was added a 100 mM Tris buffer (pH 7.0)at an amount of {fraction (1/10)} volume of the culture, and thesuspended cells were homogenated by sonicating three times for 30 secwith the aid of a sonifier, followed by centrifugation at 12000×g for 60min. The supernatants were electrophoresed on a 10% polyacrylamide gel.

The polyacrylamide gel electrophoresis showed that the levansucrase waswater-soluble by 6 hours after the IPTG induction and was graduallyconverted into a water-insoluble one from 8 hours after the IPTGinduction with a gradual decrease in enzyme activity. At 10 hours afterthe IPTG induction, the levansucrase was measured to have an enzymeactivity of 10%.

Example 2

A PCR was carried out using two primers while the plasmid pZL8 isolatedfrom E. coli KCTC 8546P served as a template. Using the PCR product, thesame procedure as Experimental Example 2 was repeated to construct theplasmid pEL12 capable of expressing the levansucrase carrying histidineresidues at its C-terminus in E. coli, which was then used for thetransformation of E. coli BL21 (DE3).

The transformed E. coli BL21 (DE3)/pEL12 was deposited in KoreanCollection for Type Cultures, Korean Research Institute of Bioscienceand Biotechnology on Apr. 25, 1995 at deposition No. KCTC 8861P.

Example 3

Ni-NTA resin (Quagen, U.S.A.) was charged in a 1.5×20 cm column throughwhich a 50 mM phosphate buffer (pH 8.0) was then flowed to give a columnfor metal ion-affinitive chromatography.

E. coli KCTC 8861P of Example 2 was cultured in the same manner as theStep 3 of Example 1 and sonicated. The cell homogenate was loaded on thecolumn which was then washed with a buffer (50 mM sodium phosphate, 300mM NaCl, 10% glycerol, pH 6.0), followed by detaching the enzyme fromthe resin by flowing a 0.3 N imidazole solution (pH 7.0). The result isgiven in FIG. 4. As shown in FIG. 4, the enzyme was purified with above95% of homogeneity (lane 2), from the total protein of E. colilysate(lane 1).

Example 4

Using the levansucrase carrying histidine residues at its C-terminal,obtained in Example 3, the experimental procedures of Examples 3 to 8were repeated. The data demonstrate that the recombinant levansucrase tothe C-end of which histidine residues were attached, has levanproduction ability almost identical to that of natural levansucrase.

Experimental Example 3 Levan Production

In 5 ml of 50 mM acetic acid buffers which were controlled to pH 3-7.5,sucrose was dissolved to the concentration of 10%, followed by theaddition of 0.42 U of the crude enzyme obtained in Experimental Example2. The resulting mixture was allowed to react at 10° C. for 5 hours and48 hours, after which a measurement was made of the amount of the levanproduced.

The results are given in Table 2, below. As apparent from the data,levan was produced in the largest quantity when the buffer was pH 5.

TABLE 2 Production Amounts of Levan According to pH of Buffer (g/l) PHTemp. 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 5 6.6 8.0 9.3 10.0 10.5 9.2 7.0 5.54.3 3.0 48 26.7 30.0 32.9 35.2 35.9 31.1 28.0 27.5 22.1 17.1

Example 5

In 10 ml of a 50 mM acetic acid buffer (pH 5.0) was dissolved sucrose tothe concentration of 10% and was added 1.05 U of the crude enzymeobtained in Experimental Example 2. The resulting mixture was allowed toreact at −3° C., 0° C., 5° C., and 10° C. for up to 200 hours, afterwhich a measurement was made of the amounts of the levan producedaccording to the condition parameters.

The results are given in Table 3, below. As apparent from the data, areaction temperature of 10° C. was the best condition for the crudeenzyme to produce levan until 50 hours of the reaction.

TABLE 3 Production Amounts of Levan According to Reaction Temp. (g/l)Time period Temp. 4 10 27 46 78 167 −3° C.  7.3 10.5 20.5 30.0 34.5 30.5 0° C. 10.1 15.7 29.5 36.5 37.2 32.3  5° C. 11.3 22.2 33.2 36.4 38.433.0 10° C. 15.2 29.3 34.9 36.7 37.5 29.7

Example 6

In 10 ml of a 50 mM acetic acid buffer (pH 5.0) was dissolved sucrose tothe concentrations of 5-40% and was added 2.08 U of the crude enzymeprepared in Experimental Example 2. The resulting mixture was allowed toreact at 10° C. for up to 60 hours, after which a measurement was madeof the amounts of the levan produced according to the conditionparameters.

The results are given in Table 4, below. As apparent from the data, thelevan amounts produced in a sucrose concentration of 30% were greaterthan in any other sucrose concentration.

TABLE 4 Production Amounts of Levan According to Sucrose Concentration(g/l) Sucrose Time period Concentration 2 11 18 25 36 59 5 12.1 19.021.8 21.6 21.4 20.8 10 14.8 38.2 36.7 39.1 34.2 34.9 20 14.1 51.4 56.752.9 51.4 52.0 30 14.6 52.4 71.1 66.9 69.8 59.4 40 11.5 39.1 59.8 58.860.0 56.4

Example 7

In 5 ml of a 50 mM acetic acid buffer (pH 5.0) was dissolved sucrose tothe concentration of 10% and were added 0.42 U, 1.05 U, and 2.08 U ofthe crude enzyme prepared in Experimental Example 2. The resultingmixtures were allowed to react at 10° C. for up to 150 hours, afterwhich a measurement was made of the amounts of the levan producedaccording to the condition parameters.

The results are given in Table 5, below. It is apparent from the datathat the levan is produced at larger amounts by the greater activity ofthe enzyme.

TABLE 5 Production Amounts of Levan According to Enzyme Concentration(g/l) Enzyme Time period Concentration 4 12 48 96 144 0.42 U 2.5 4.021.7 35.7 39.0 10.5 U 4.9 12.9 34.9 48.5 42.2 20.8 U 24.9 44.9 49.0 49.246.2

Example 8

In 5 ml of a 50 mM acetic acid buffer (pH 5.0) was dissolved sucrose tothe concentration of 20% and were added 2.08 U of the crude enzymeprepared in Experimental Example 2. The resulting mixtures were allowedto react at 10° C. for up to 36 hours, after which a measurement wasmade of the amounts of the levan and other saccharides producedaccording to the condition parameter.

The results are given in Table 6, below. It is apparent from the datathat the levan is produced at larger amounts by the greater activity ofthe enzyme.

TABLE 6 Production Amounts of Levan According to Reaction Time (g/l)Time period Saccharides 0 6 11 18 25 36 Levan 0 28.3 51.4 56.7 52.9 51.4Residual Sucrose 200 161 125 120 98 10 Glucose 0 237 36.0 50.4 74.6104.8 Fructose 0 2.5 5.8 6.2 6.5 7.2

Example 9

In 10 ml of a 50 mM acetic acid buffer (pH 5.0) was dissolved raw sugar(DaeHan Sugar Co.) to the concentration of 20% and were added 1.05 U ofthe crude enzyme prepared in Experimental Example 2. The resultingmixtures were allowed to react at 10° C. for up to about 91 hours, afterwhich a measurement was made of the amounts of the levan and othersaccharides produced. Levan was produced at an amount of 12.5 g/l after11 hours of the reaction, 25.0 g/l after 25 hours, and 45.8 g/l after 91hours.

Experimental Example 4

Selection of Strains Containing Levan Fructotransferase Capable ofProducing DFA IV from Levan

Strains were selected from samples taken from soil in Korea. 0.5 gramsof each of the soil samples was suspended in 9.5 ml of distilled waterand the suspensions were successively diluted to 10-fold, 100-fold and1000-fold. 200 ul of each of the dilutions was spread on the surface ofa selective agar plate, and incubated at 30° C. for 72 hours in anincubator. The selective agar medium contained 0.5% (w/v) of levan as amajor carbon source, along with 0.3% of NaNO₃, 0.05% of MgSO₄, 0.02% ofMnCl₂, 0.1% of K₂HPO₄ and 1.5 agar/L. A solution of a predeterminedamount of levan in water was passed through a 0.45 micron filter and thefiltrate was mixed with the other nutrients before being added to asterilized medium. After the incubation, colonies around which halos hadbeen formed were primarily selected as being capable of decomposinglevan.

Inocula picked from the primarily selected colonies were inoculated inselective broths, each containing levan as a major carbon source, andcultured at 30° C. for 72 hours in an incubator. Investigation was madeinto the growth of the strains inoculated and the metabolites resultingfrom the metabolism of levan through thin layer chromatography (TLC). Ofthe 250 strains primarily selected, 8 strains of microbes were found toproduce difructose dianhydride. Finally, one strain which stablyproduced difructose from levan with high enzyme activity was selectedand named “K2032”.

Identification of Strain K2032

The identification of the selected strain K2032 was achieved byexamining its biochemical characteristics and the composition andcontent of its fatty acids and quinones and comparing them with thedatabase previously accumulated. Many experiments revealed that strainK2032 is Gram-variable and is able to utilize glucose, fructose,galactose, arabinose, mannitol, xylose, ribose, sorbitol, cellobiose,glycogen, sucrose, acetic acid, propionic acid, and salicin, but notrhamnose, malonate, adipic acid, N-acetyl glutamic acid, and adipicacid. As for its intracellular fatty acid composition, the bacteriacomprises C15 anteiso/C17 anteiso/C16 iso at a ratio of 51% /14.5%/3.6%with C16/C14 being at a ratio of 17.8%/7.1%. As a result of the analysisof quinones, which are important components for the chemotaxamomy ofmicrobes, the bacteria contained menaquionone MK-9(H₂).

With the analysis results, the strain K2032 was identified asArthrobacter ureafaciens, and thus, named A. ureafaciens K2032 (Table7).

TABLE 7 Phenotypic and chemotaxonomic characteristics of strain K2032variable Gram reaction (+ when young) Acid from glucose − Nitratereduction − Starch hydrolysis − Growth in 10% NaCl − Utilization ofD-glucose, D-fructose, D-galactose, + L-arabinose, D-mannitol, D-xylose,D-ribose D-sorbitol, cellobiose, glycogen, sucrose, acetic acid,propionic acid, salicin Utilization of L-rhamnose, malonate, adipicacid, − N-acetyl glutamic acid, L-serine Cellular fatty acid; C15anteiso/C17 anteiso/C16 iso 51%/14.5%/3.6% C16/C14 17.8%/7.1% Majormenaquinone MK-9(H₂)

Experimental Example 5

Purification of Levan Fructotransferase from Arthrobacter

A. ureafaciens K2032 was inoculated in a selective broth containinglevan as a major carbon source and cultured at 30° C. for 72 hours in anincubator. Investigation into the growth and levan fructotransferaseactivity of the bacteria provided the knowledge that the largest enzymeactivity is obtained after 10 hours of the incubation. From the cultureincubated for 10 hours, the enzyme was purified (FIG. 5). In thisregard, the purification was achieved by successively using acetoneprecipitation, ion exchange chromatography (DEAE 650-M and Mono Q), andgel filtration chromatography, resulting in producing levanfructotransferase with a specific activity of 2269 U/mg protein at anamount of 1.1 mg at a yield of 29.8% (Table 8).

TABLE 8 Summary of levan fructotransferase purification from A.ureafaciens K2032. Total Spec. Act. Purifi- Purification Volume activityProtein (U mg⁻¹) cation Yield step (ml) (U) (mg) protein) Fold (%)Culture 3,800 8,360 182.3 45.9 1.0 100 supernatant Acetone 700 5,67071.3 79.5 1.7 67.8 precipitation Ion-exchange 5.5 4,821 5.0 964.2 21.157.7 adsorption 2^(nd) Ion- 3.2 3,416 2.6 1313.8 29.2 40.9 exchangeadsorption Gel permeation 1.2 2,496 1.1 2269.0 49.8 29.8 (Superose 12)Characterization of the Purified Enzyme

The purified enzyme was 51,000 in molecular weight as measured bySDS-PAGE while being measured to have a molecular weight of 96,000 bygel filtration chromatography. Thus, it was identified as being a dimerin aqueous conditions. An amino acid analysis showed the amino acidsequence of the C-terminus of the enzyme. Maintaining its stability in apH range of 4-10.5, the enzyme showed optimal activity at around pH 6.0.Also, it was of optimal activity at 55° C. After being allowed to standat 50° C. for 30 min, the enzyme was observed to have a remnant activityof 90% or greater (Table 9). Whereas being inhibited by Mn²⁺, Fe²⁺ andHg²⁺, the activity of the enzyme was enhanced by Na²⁺ and Ca²⁺.

TABLE 9 Effect of additives on the activity of levan fructotransferasefrom A. ureafaciens K2032 Concentration Relative activity Additive (mM)(%) Control — 100 NaCl 1, 10 125, 142 NH₄Cl 1  46 MnCl₂ 1  9 CaCl₂ 1, 10156, 181 NiCl 1  74 ZnCl₂ 1  23 LiCl 1  92 MgCl₂ 1  63 FeCl₂ 1  4 HgCl₂1  0 KCl 1  89 EDTA 1, 10  95, 94 SDS 1, 10 101, 32

Experimental Example 6

Isolation of Genomic DNA from Arthrobacter

Isolation of a gene coding for a levan fructotransferase from A.ureafaciens was performed using a genome DNA isolation kit, such as thatsold by Bio 101, identified as “G NOME™ DNA ISOLATION KIT”, according toits instruction. First, A. ureafaciens K2032 was inoculated in 50 ml ofa selective broth and cultured for 24 hours, followed by centrifugationat 3,000 rpm for 5 min to harvest cells. To the cell pellet was addedthe cell suspension solution (10 mM Tris-HCl (pH 8.0), 0.1 M EDTA (pH8.0)) to the volume of 1.85 ml. This cell suspension was well mixed with100 ul of the cell lysis/denaturing solution (0.5% SDS), along with 50ul of the RNase solution, and incubated at 55° C. for 15 min. To removeproteins from the sample, 25 ul of the protease was added and incubatedat 55° C. for 1 hour. After 500 ul of the salt out solution was added,the mixture was aliquoted in 1.5 ml tubes which were then cooled at 4°C. for 10 min. Following centrifugation at 12000 rpm for 10 min, thesupernatant was transferred to a 15 ml tube, in which 2 ml of TE bufferand 8 ml of 100% ethanol were added to induce DNA precipitation. The DNAprecipitates were collected by centrifugation at 12,000 rpm for 15 min,dried in the air and dissolved in a TE buffer.

Cloning of Levan Fructotransferase-Coding Gene

The genome DNA prepared from A. ureafaciens was digested with variousrestriction enzymes and electrophoresed on 1% agarose gel, after whichDNA fragments in a size range of 3-4 kb were eluted from the agarosegel. The partial genomic DNA fragments were ligated into the cloningvector pBluescript KSII⁺ which was previously cut with BamHI. Thisrecombinant plasmid was transformed into E. coli DH5α. Of thetransformants, the cells carrying a gene coding for a levanfructotransferase were selected as follows.

First, a sequence in the N-terminal amino acid sequence of the K2032strain and a second amino acid sequence, which was found to be in ahomology relationship with levan fructotransferase, were used tosynthesize degenerated primers. Using these primers, a standard PCR wascarried out with the genomic DNA of K2032 bacteria serving as atemplate. The PCR products ranging, in size, from 600 to 650 bp wereeluted from the gel and introduced into shuttle vectors for E. coli. Asa result of restriction enzyme gene mapping, the DNA fragmentsintroduced were identified as being divided into four kinds and thevectors carrying these DNA fragments were called pDA11, 17, 18 and c8,respectively. In addition, base sequencing analysis of the four DNAfragments showed that the DNA fragment inserted in pDA11 and the levanfructotransferase gene of A. nicotinovorans are in high homology (85%)relationship. Therefore, the DNA fragment introduced in pDA18 could beused as a probe for the Southern hybridization with the DNA preparedfrom A. ureafaciens K2032 with the aim of detecting the levanfructotransferase gene anchored in the bacteria. Before the Southernhybridization, the genomic DNA of A. ureafaciens K2032 was digested withvarious restriction enzymes. As a result of the Southern hybridization,a signal was detected in a 5.6 kb DNA fragment upon digestion with ClaI,in a 8.0 kb DNA fragment upon digestion with PstI, and a DNA fragmentlonger than 10 kb upon digestion with BamHI. Accordingly, the genomicDNA was cut with either ClaI or PstI and electrophoresed on a 1% agarosegel, after which DNA fragments in a size range of 5-10 kb were elutedfrom the agarose gel. The partial genomic DNA fragments were ligatedinto the cloning vector pBluescript KSII⁺ which was previously cut withClaI or PstI. These recombinant plasmids were transformed into E. coliDH5α. Of the transformants, the cells carrying a gene coding for a levanfructotransferase were selected by Southern hybridization. Finally, whencutting with ClaI, there was obtained plasmid pDC a carrying a 5.6 kbDNA fragment. On the other hand, when cutting with PstI, there wasobtained plasmid pDpst carrying a 8.0 kb DNA fragment.

Isolation Analysis and Subcloning of the Recombinant Plasmids

DNA preparation from the transformed E. coli was carried out by anordinary boiling method and with the aid of a commercially availablekit. To analyze the recombinant plasmids, they were digested withrestriction enzymes and electrophoresed on 1% agarose gel.

The restriction enzyme gene mapping of the DNA fragments introduced inpDcla and pDpst, the analysis of gene loci, and the Southern blottingwith DNA fragments showed that pDcla and pDpst carried an N-terminalhalf and a C-terminal half of the gene of interest with an overlappingstretch of about 200 bp therebetween. No enzymatic activity was detectedfrom the bacteria which anchored the two plasmids respectively.According to a base sequencing analysis, the sites for restrictionenzymes PstI, ApaI and NotI were found to be present in both of theplasmids. By taking advantage of these common restriction enzyme sites,there was constructed plasmid pDF8 which has a complete open readingfame (ORF). A DNA fragment 3.5 kb long was introduced in the plasmidpDF8 and enzymatic activity was detected from the bacteria anchoringthis plasmid.

Base Sequence of the Cloned Levan Fructotransferase Gene and ItsPutative Amino Acid Sequence

In order to analyze the nucleic acid base sequence of the 3.5 kb DNAintroduced into pDF8, DNA ladder fragments which showed a regulardistance of about 300-400 bp were made and subjected to a sequencingprocess. As a result, a sequence of 3345 bases was ascertained and foundto have only one ORF. Using the BLAST search, which is available fromthe Internet, the ORF was investigated for homology. In result, a levanfructotransferase gene was detected as being high in the homology withthe ORF, and thus, it was named lftA. FIG. 6 shows the base sequence oflftA and the amino acid sequence deduced therefrom.

The newly isolated levan fructotransferase structural gene is composedof 1566 bp which corresponds to 521 aa. The gene of the presentinvention has a signal sequence consisting of 99 bp (corresponding to 33aa) at its N-terminus and therefore, the putative completed protein maybe about 56 kDa in molecular weight. Its isoelectric point was found tobe pH 5.15. This levan fructotransferase gene is longer by 15 bases (5amino acids) than is the levan fructotransferase gene derived from A.nicotinovorans GS-9. These two genes are 81% in the homology of aminoacid sequence (76% in the homology of base sequence). Consequently, thelevan fructotransferase gene of A. ureafaciens K2032 was identified asbeing new and its base sequence was registered in the GenBank, U.S.A.(Accession No. AF181254).

Experimental Example 7

Development of Highly Expressable, Transformed E. coli CarryingRecombinant Levan Fructotransferase Gene

Using the restriction enzyme PstI, the lftA gene was cut at its signalsequence, followed by Klenow treatment to give a blunt end. The lftAgene was divided by cutting with NotI. The Klenow-treated DNA fragmentwas further digested with SmaI and ligated to the pUC118 vector whichwas previously treated with CIP, so as to construct pUDFA18. This wasintroduced into E. coli DH5α. FIG. 7 shows the plasmid pUDFA18 which isobtained by inserting the lftA gene in the expression vector pUC118.FIG. 8 shows the total protein pattern of E. Coli JUD81 in SDS-PAGE. TheE. coli JUD81 is the E. coli DH5 transformed with pUDFA18.

Experimental Example 8

Production of DFA IV from Levan by Use of Recombinant LevanFructotransferase

In 1,000 ml of warm water was dissolved 20 g of levan, cooled andcontrolled to pH 6.5 with a phosphate buffer. To the resulting solution,E. coli JUD81 homogenate was added and allowed to react at 37° C. FIG. 9shows the change of the amounts of DFA IV and other saccharides in thereaction mixture in accordance with reaction time period. After 40 hoursof the reaction, the reaction mixture was allowed to stand in hot waterfor 5 min to inactivate the remaining enzyme and loaded onto a charcoalcolumn (diameter 6.5 cm, height 20 cm). The column was washed withdistilled water until no saccharides were detected in the washings. Thecolumn was again washed with 1,000 ml of 5% ethanol and then with 1,000ml of 25% ethanol to elute the absorbed DFA IV. The effluents containingDFA IV were collected and concentrated to a volume of 30 ml by use of arotary evaporator. To the concentrate, 100% ethanol was added to thefinal ethanol concentration of 95% or higher, so as to crystallize DFAIV. The DFA IV precipitates were washed many times with pure ethanol anddried to yield 2.5 g of pure DFA IV.

INDUSTRIAL APPLICABILITY

The analysis data from NMR, HPLC and TLC after acidolysis demonstratethat the DFA IV obtained is identical to a standard sample. FIG. 10 is aNMR result for the DFA IV obtained.

The present invention has been described in an illustrative manner, andit is to be understood that the terminology used is intended to be inthe nature of description rather than of limitation. Many modificationsand variations of the present invention are possible in light of theabove teachings. Therefore, it is to be understood that within the scopeof the appended claims, the invention may be practiced otherwise than asspecifically described.

1. A polynucleotide of SEQ ID NO: 2 encoding for a levanfructotransferase of the amino acid SEQ ID NO: 1 isolated fromArthrobacter ureafaciens K2032, which can hydrolyze levan to producedifructose dianhydride IV.
 2. A recombinant expression vector pUDFA81carrying the polynucleotide sequence of SEQ ID NO:
 2. 3. An organismEscherichia coli JUD81 KCTC 0877BP which is prepared by transformingEscherichia coli DH5α with the expression vector pUDFA81.